Vol. 2: 143–151, 2008 AQUATIC BIOLOGY Printed June 2008 doi: 10.3354/ab00046 Aquat Biol Published online May 13, 2008

OPENPEN ACCESSCCESS Ultrastructural studies of oogenesis in the variable abalone Haliotis varia (Vetigastropoda: Haliotidae)

T. M. Najmudeen*

Department of Marine Biology, Microbiology and Biochemistry, School of Ocean Science and Technology, Cochin University of Science and Technology, Cochin-16, India

ABSTRACT: Ultrastructural features of the ovary as well as oogenesis were examined in the variable abalone Haliotis varia with the help of transmission electron microscopy. Based on the ultrastructure, the female germ cells were divided into 5 stages; oogonia, pre-vitellogenic, early vitellogenic, late vitellogenic and mature oocytes. Oogonia were supported by follicle cells that lined up the inner walls of the ovary. Vitellogenesis in H. varia commenced at about 50 µm oocyte diameter, with a major contribution by the autosynthetic pathway. Lipid droplets and protein yolk granules, measur- ing 2.3 ± 0.2 and 2.5 ± 0.3 µm, respectively, were observed in late vitellogenic and mature oocytes. Vitellogenic oocytes were encircled by a vitelline envelope consisting of a single layer of about 2.3 µm thickness. Numerous microvilli were embedded in the vitelline envelope. Cytoplasmic inclu- sions such as cortical granules, lipid droplets, mitochondria and yolk granules constituted the funda- mental components of the mature oocyte. The cortical granules were arranged beneath the oolemma at the cortex of the oocyte. Oocyte degeneration in H. varia was observed in all stages of oocyte development, except in oogonial cells and pre-vitellogenic oocytes.

KEY WORDS: Female germ cells · Haliotis varia · Ovarian maturation · Variable abalone · Ultrastructural studies · Vitellogenesis

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INTRODUCTION Whilst the gametogenic cycle and gonad histology of various abalone species have been extensively stud- Vitellogenesis in marine molluscs is a complex pro- ied (reviewed by Hahn 1989), there is less literature on cess involving both autosynthetic and heterosynthetic the ultrastructural changes taking place in their germ pathways that contribute to the accumulation of more cells. Most of the investigations along this line have than one type of storage product (Eckelbarger & focused on the fine-structure analysis of spermatozoa Young 1997, Dreon et al. 2002, Pal & Hodgson 2002, and cytochemistry of acrosomal vesicles (e.g. Healy et Matsumoto et al. 2003). Various kinds of yolk compo- al. 1998, Galindo et al. 2003). Several authors have nents in this group may be formed in many different classified the female germ cells of abalone, in both ways, directly or indirectly, from the Golgi bodies, temperate and tropical habitats (Young & DeMartini mitochondria, or nucleolar material, or independently 1970, Takashima et al. 1978, Martin et al. 1983). in the cytoplasm (Eckelbarger & Davis 1996, Osada et Nevertheless, there is no consensus in assigning the al. 2003). In some groups, there is strong evidence for number of stages to ovarian cells, and most of these the involvement of extra ovarian components in vitel- studies have been based on the histological examina- logenesis (Amor et al. 2004). Many authors have dis- tion and subsequent structural features obtained. Clas- cussed the role of ovarian ultrastructure and the mech- sification of ovarian cells based on ultramicroscopic anisms of vitellogenesis on the evolutionary forces that observations is comparatively rare, and the available shaped the life-history patterns of marine gastropods literature only describes the oogonial cells in detail (Eckelbarger 1994, Hodgson & Eckelbarger 2000, (Martin et al. 1983, Sobhon et al. 1999, Apisawetakan Hodgson et al. 2002, Pal & Hodgson 2002, Pal 2007). et al. 2001).

*Email: [email protected] © Inter-Research 2008 · www.int-res.com 144 Aquat Biol 2: 143–151, 2008

Haliotis varia, one of the least exploited species of washed 3 times in cacodylate buffer for about 15 min tropical abalone, is distributed in India along the Gulf each time and post-fixed in 1% buffered osmium of Mannar on the east coast, and at Andaman and tetroxide for about 1 h at 4°C. Properly fixed tissues Nicobar Islands. It is a small tropical species, attaining were again washed in cacodylate buffer and then a maximum shell length of 8 cm (Wilson 1993). There is dehydrated in a graded acetone series. The tissues no report of commercial exploitation of this species in were infiltrated and embedded in a low-viscosity, India, which is attributed to its small size and limited resin-embedding medium (Spurr 1969) and kept at distribution. However, recent increase in the demand 60°C for 36 h. Ultra-thin sections (50 to 60 nm) were cut for small cocktail size abalone in many Asian markets from the polymerized blocks using a glass knife on an (Guo et al. 1999) has created interest in developing ultramicrotome (LKB BROMA). The sections collected methods for the artificial propagation of this species. on uncoated copper grids were stained in 1% uranyl Some aspects of its reproductive biology and larval acetate in methanol and lead citrate. After drying, the rearing have been investigated recently (Najmudeen grids were examined under a Hitachi 600 electron & Victor 2004a,b, Najmudeen 2007a,b). Studies on the microscope at 50 kV and relevant areas were photo- annual reproductive cycle of this species in India con- graphed using Graphic Kodak lithe film. firm that the peak breeding season extends from December to March, with asynchronous spawning by individuals (Najmudeen 2007b). All gonad maturity RESULTS stages were found throughout the year, suggesting that gametogenesis is a continuous process. Bussara- Major changes during ovary maturation in Haliotis wit et al. (1990) also proposed greater gametogenic varia were the formation of a germinal vesicle and of activity in this species, based on their observations of yolk granules and, finally, oocyte maturation. Based on rapid post-spawning recovery at Phuket, Thailand. the changes in size and cytoplasmic contents of oocytes, Except for these reports, there is no published informa- 5 stages of oocytes were distinguished in H. varia, tion on the reproductive biology of this species. In which are dealt with in detail in the following sections. India, gonad maturation was associated with variation in water temperature and salinity; the seasonal low values coincided with the breeding season of H. varia Oogonia (Najmudeen & Victor 2004b). Since the determination of gonad maturity stages using histological examina- In the ovary, the germinal epithelium lined the inner tion is inadequate, in most cases, to identify the gamete ovarian wall. Cells in the germinal epithelium were ini- stages involved and to delineate the exact process of tially cuboidal and as they enlarged, became oval or vitellogenesis, it is important to carry out an ultrastruc- round oogonia. The oogonial cells in the ovary were sup- tural study of the gonad. In this perspective, the pre- ported by follicle cells, which lined up the perpendicu- sent paper examines the ultrastructural changes of the larly running trabecular network (Fig. 1A). Oogonia had female germ cells during maturation in the variable a diameter of 10 ± 2 µm (±SD, n = 30) and were domi- abalone H. varia based on transmission electron nated by a large ovoid nucleus of 9 ± 2.5 µm (±SD, microscopy (TEM), to describe the process of vitelloge- n = 30) diameter (Fig. 1B). The cytoplasm of oogonia con- nesis and to supplement additional information for the tained numerous free ribosomes, mitochondria and some classification of female germ cells. clusters of electron dense granules. Attached to the base of each oogonial cell were a few small follicle cells (Fig. 1C). The nucleus of oogonial cells contained less- MATERIALS AND METHODS condensed chromatin materials scattered in the nucleo- plasm. Nucleoli were not conspicuous at any stage of Adult specimens of female Haliotis varia were col- oogonia, and the oolemma appeared smooth and without lected from the intertidal rocks at Tuticorin Harbour any particular morphological specialization at this stage. basin of the Gulf of Mannar, southeast coast of India (08°45’ N, 78°12’ E) from a depth of 2 m in January and February 2000. Immediately after collection, pieces of Pre-vitellogenic oocytes ovary from 20 animals (size range from 35 to 56 mm shell length) were separated from the digestive gland The pre-vitellogenic oocytes of Haliotis varia were tissue and placed in fresh cold (4°C) primary fixative of more elongated than the oogonia and measured 16 ± 3% gluteraldehyde buffered with 0.2 M cacodylate 2 µm (±SD, n = 30) in diameter along their long axis containing 2% tannic acid and 6% glucose at pH 7.2, (Fig. 1D). Nuclear volume increased, which measured for 3 h. For TEM, fragments of selected material were 12 ± 2 µm (±SD, n = 30) in diameter. As the pre-vitel- Najmudeen: Ultrastructure of oogenesis in Haliotis varia 145

Fig. 1. Haliotis varia. TEM of developing ovary. (A) Ultrastructure of ovarian lumen (ol) showing the arrangement of follicle cells (fc) along the trabecular network (tb).(B) Fine structure of the oogonium with a well-defined nucleus (n) containing chromatin bodies (ch) and a nuclear membrane (nm); cytoplasm (c) contains numerous ribosomes (r) and mitochondria (m). (C) Enlarged view of oogonium showing granular nuclear emissions (ne), chromatin bodies and nuclear membrane. (D) Early vitellogenic oocyte with an enlarged clear nucleus and darkly stained nucleolus (nl), and lipid droplets (li) in the cytoplasm. (E) Early vitello- genic oocyte with increased cytoplasmic area and electron lucent space (es) in the nucleolus. (F) Enlarged view of an early vitello- genic oocyte showing the nuclear pores (np) and nuclear emissions (ne) 146 Aquat Biol 2: 143–151, 2008

logenic oocytes became larger, they moved away from Concentric and closed layers of smooth ER encompass- the germinal epithelium and advanced towards the ing several granules and vacuoles were discernible in digestive gland, but remained attached by stalks. The the vicinity of the nucleus. These were identified as cytoplasm became strongly basophilic, indicating con- membrane specializations and might be associated siderable protein synthesis. As the enlargement of the with the synthesis of yolk materials (Fig. 2E). As vitello- oocyte progressed, the nuclear:cytoplasmic ratio of the genesis progressed, these membrane specializations oocyte decreased. The nucleoplasm contained less- developed considerably, often encompassing several dense chromatin materials. A prominent electron vesicles and yolk materials. Lipid droplets in the cyto- dense nucleolus was discernible in the nucleus of the plasm measured about 2.3 ± 0.2 µm (±SD, n = 30) in oocyte at this stage. Numerous gaps or nuclear pores diameter, and protein yolk vesicles measured about were evident in the nuclear wall, around which elec- 2.5 ± 0.2 µm (±SD, n = 30) in diameter (Fig. 2D). Other tron dense granular materials were clumped together lamellar structures observed during vitellogenesis were (Fig. 1D). The cytoplasm of developing oocytes con- annulate lamellae, which also originated in close tained many ribosomes, mitochondria, Golgi bodies, as proximity to the nucleus (Fig. 2F). well as rough endoplasmic reticulum (ER) indicating active synthesis of protein. Mature oocytes

Early vitellogenic oocytes Mature oocytes of Haliotis varia measured 170 ± 30 µm (± SD, n = 30) in diameter. A vitelline envelope At about 50 µm diameter, small, lightly stained appeared around the oolemma of mature oocytes, spherical bodies appeared in the cytoplasm of the which measured 2.3 ± 0.2 µm (±SD, n = 30) in thickness developing oocytes (Fig. 1E). This stage indicated the (Fig. 3A,B). Numerous microvilli were embedded in onset of yolk synthesis, i.e. vitellogenesis. Less electron the vitelline envelope, arranged about 0.2 µm apart. dense, round lipid droplets appeared in the cytoplasm The free ends of the microvilli protruded out to the of early vitellogenic oocytes. Several cell organelles vitelline envelope. The nucleus was very large and less were present in the cytoplasm of the oocyte, which electron dense with a dark nucleolus. The cytoplasm might be associated with the formation of yolk compo- contained numerous lipid droplets and protein yolk nents. No pinocytes were seen in the early vitellogenic granules. Cortical granules, the membrane-bound oocytes. The nucleus of early vitellogenic oocytes en- vesicles, were seen in mature oocytes (Fig. 3B). They compassed a clear round space (Fig. 1F). Large num- were round with a diameter of about 0.3 µm and occu- bers of ribosome-studded, as well as smooth, mem- pied a layer beneath the cytoplasmic membrane of the brane-bounded vesicles were also present in the mature oocyte. Other types of cell organelles were not cytoplasm of early vitellogenic oocytes (Fig. 2A). observed in the cytoplasm at this stage. At the final stage of oocyte maturation, the oocytes became detached from the trabeculae and tightly packed in the Late vitellogenic oocytes ovarian lumen.

Cytoplasm of late vitellogenic oocytes was uniformly filled with various quantities of darkly stained protein Degenerating oocytes yolk granules and lipid droplets (Fig. 2B). Small granu- lar materials were also seen in the cytoplasm. The Necrosis of vitellogenic oocytes was observed in nucleus occupied a very large volume of the cytoplasm vitellogenic and mature oocyte stages. The first visible and migrated to the free end of the oocyte. The nucle- sign of necrosis occurred in the nucleus. The chro- olus was a dark, round, electron-dense body in the matin became less granular; the nuclear membrane nucleoplasm. The late vitellogenic oocytes of Haliotis became convoluted and broke down. Numerous vacu- varia measured 80 to 120 µm (n = 60) in diameter and oles appeared in the ooplasm, and the perivitelline were attached to the trabeculae in the ovarian lumen space between the oolemma and vitelline envelope near the digestive gland. Large numbers of late vitel- increased (Fig. 3C). The microvilli were separated logenic oocytes were found in the late maturing stage from the oocyte surface, and finally the vitelline enve- ovaries of H. varia. lope disintegrated (Fig. 3D). The yolk vesicles were Concentric layers of rough ER were present sur- encircled by autophagic vacuoles. Genital blood ves- rounding the lipid droplets (Fig. 2C), indicating their sels were also present near the degenerating oocytes role in lipid yolk synthesis. Numerous mitochondria (Fig. 3E). Early vitellogenic oocytes were also subject and free ribosomes were also present in the cytoplasm. to degeneration. Najmudeen: Ultrastructure of oogenesis in Haliotis varia 147

Fig. 2. Haliotis varia. TEM of developing and mature ovaries. (A) Cytoplasm of early viteloogenic oocyte that has initiated the synthesis of lipid yolk bodies (ly) and has numerous mitochondria (m), ribosomes (r) and a well-defined oolemma (o). c: cyto- plasm; n: nucleus. (B) Late vitellogenic oocyte with both lipid yolk and protein yolk (py), and granular materials (gr). (C) Mem- brane specializations in the late vitellogenic oocyte encompassing lipid yolk components made up of rough endoplasmic reticu- lum (rer) containing ribosomes. (D) Cytoplasm of mature oocyte with its fundamental components: lipid yolk, protein yolk, mitochondria and endoplasmic reticulum (er). (E) Membrane specializations in the late vitellogenic oocyte that are formed by smooth endoplasmic reticulum (ser) and granular materials (g); some vacuoles (v) can also be seen. (F) Annulate lamellae in the cytoplasm of a late vitellogenic oocyte made up of rough endoplasmic reticulum and in close proximity to both lipid yolk and protein yolk 148 Aquat Biol 2: 143–151, 2008

Fig. 3. Haliotis varia. TEM of mature and disintegrating oocytes. (A) Periphery of the mature oocyte with vitelline envelope (vm) containing numerous microvilli (mv). Cyto- plasm contains numerous lipid yolk (ly) and protein yolk (py) bodies and coritical granules (cg). Note perivitelline space (ps). (B) Enlarged view of a vitelline envelope of a ma- ture oocyte. Cortical granules are round bodies arranged beneath the oolemma. (C) Degenerating oocyte of H. varia showing the partly detached cytoplasmic contents from the oolemma (ol). Some vacuoles (v) are also present. (D) De- generating oocyte with a disintegrating vitelline envelope (dvm) and a greater number of autophagic vacuoles (av) and disintegrating yolk granules (dyg). (E) Ultrastructure of ovarian lumen with degenerating oocytes (do)

DISCUSSION mitotic multiplication. In the vegetative phase of devel- opment, oocyte growth is very considerable, princi- Oogenesis in molluscs can be divided into a genera- pally due to vitellogenesis, and requires substantial tive (proliferative) and vegetative (growth) phase (An- uptake and synthesis of nutritive materials (Pipe 1987). derson 1974). During the proliferative phase, the num- The oogonial cells of Haliotis varia described in the ber of oogonial cells increases in the germinal zone by present study were similar to the description given to Najmudeen: Ultrastructure of oogenesis in Haliotis varia 149

the oogonial cells of donkey’s ear abalone H. asinina is also an indication of increased involvement of intra- by Apisawetakan et al. (2001). The nucleus of the oocytic components in the fabrication of yolk compo- oogonial cells contained less condensed chromatin nents in H. varia. material scattered in the cytoplasm. Numerous ribo- Various cell organelles, such as the ER, Golgi bodies, somes, mitochondria and lipid inclusions appeared in mitochondria, autophagous vacuoles and multivesicu- the cytoplasm of oogonial cells. Similarly, some irregu- lar bodies have been assumed to be involved in the for- larly shaped lipid inclusions along with ribosomes and mation of yolk granules in molluscan oocytes (Eckel- mitochondria were reported in the oogonial cells of barger & Davis 1996). It is apparently the combined many other marine molluscs (Hodgson & Eckelbarger activity of a number of these organelles that leads to 2000, Pal & Hodgson 2002). The oogonial cells of the formation of the complex proteinaceous yolk gran- H. varia were lined up within or in close vicinity of the ules. Various membrane specializations comprised of germinal epithelium. rough and smooth endoplasmic reticulum were The most prominent feature of the undifferentiated observed in the developing oocyte of Haliotis varia. oocytes of Haliotis varia was the large nucleus, which The concentric membrane specializations encompass- occupied more than half of the diameter of the cell. ing several vesicles were seen in the developing One of the first indications that an oogonium is differ- oocyte. This may be identical to Balbiani’s vitelline entiating into an oocyte is the appearance of charac- body, found in the oocytes of many molluscs, which teristic pores in the nuclear membrane (Verhey & plays a role in vitellogenesis (Gómez-Robles et al. Moyer 1967). Electron dense nucleolar materials were 2005). Balbiani’s vitelline body, which occurs in devel- clumped together in close proximity to the nuclear oping oocytes of a wide spectrum of species through- pores in the primary oocytes of H. varia. In H. asinina, out the animal kingdom (Guraya 1979), was also these materials were categorised into ribosome-linked observed in the vitellogenic oocytes of the bivalve particles around the cytoplasmic side of the nuclear Mytilus edulis (Pipe 1987). Similar types of membrane membrane (Apisawetakan et al. 2001). These electron specializations are distributed in the vitellogenic dense granular materials were clumped near the outer oocytes of the neogastropod Bolinus brandaris (Amor nuclear membrane in H. varia. During the early vitello- et al. 2004). These cell organelles and membrane spe- genic phase, the cytoplasm became strongly baso- cializations were in close proximity to the yolk granule philic, indicating considerable synthesis of protein. in the vitellogenic oocytes of H. varia. RNA materials are transported from the nucleus to the In addition to the autosynthetic process, there are cytoplasm prior to the onset of vitellogenesis. Adiyodi possibilities for heterosynthetic accumulation of yolk & Subramoniam (1983) reported that such nuclear materials in Haliotis varia. The mechanism of transport emissions are not uncommon in the growing oocytes of of large molecules themselves through membranes is various animal groups and concluded that the transfer described in many species (Selman & Arnold 1977, of nucleolar materials to the ooplasm was a prelude to Beninger et al. 2003). They suggest that yolk precur- protein synthesis. sors may be synthesized by the RER in the syncytial Vitellogenesis is auto- and/or heterosynthetic in dif- cytoplasm, and the passage of these yolk precursors ferent molluscan species (Eckelbarger & Young 1997, through the syncytial plasma membrane and oolemma Matsumoto et al. 2003, Amor et al. 2004). In Haliotis by this mechanism would go undetected in electron varia, the primary oocytes entered into the vitellogenic micrographs. Using molecular characterization of phase at a diameter of about 50 µm, resulting in reduc- cDNA, Matsumoto et al. (2003) concluded that the tion of the basophilic nature of the ooplasm. The pro- putative vitellogenin in the Pacific oyster Crassostrea cess of vitellogenesis in different bivalves and gas- gigas is originally synthesized in the follicle cells. tropods has been described by various workers (Pipe Another important component of the proteinaceous 1987, Eckelbarger & Davis 1996, Pal & Hodgson 2002, yolk in gastropods, ferritin, is thought to be derived Amor et al. 2004). The present investigation using from the digestive gland (Bottke & Tiedtke 1988, TEM provides evidence for the formation of yolk com- Andrews et al. 1992). As in other gastropods, yolk syn- ponents via the autosynthetic pathway. The occur- thesis in H. varia involves the direct incorporation of rence of pinocytotic vesicles along the plasma mem- extraoocytic precursors from the neighboring tissues to brane of the oocyte has been taken as an important the intra-cellularly synthesized yolk vesicles. There criterion for exogenous vitellogenesis, as it indicates was a significant negative correlation between bio- the uptake of macromolecules (Jong-Brink et al. 1983). chemical composition of the digestive gland and ovary, In molluscs, morphological evidence for uptake of especially lipids and carbohydrates, in connection with macromolecules is presented for a number of species the maturation process in H. varia, indicating the (Bottke & Tiedtke 1988, Eckelbarger & Davis 1996). transfer of these biochemical components from the The lack of micropinocytotic vesicles on the oolemma digestive gland to the ovary (Najmudeen 2007a). 150 Aquat Biol 2: 143–151, 2008

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Editorial responsibility: Roderick Nigel Finn, Submitted: October 10, 2007; Accepted: April 4, 2008 Bergen, Norway Proofs received from author(s): April 29, 2008